The main objectives of these studies are (i) to identify and characterize all proteins that are specified by the defective human parvoviruses (AAV) and to determine similarities and differences with autonomous parvovirus proteins, (ii) to define the mechanism(s) by which the AAV proteins arise and (iii) to define specific functions of the AAV proteins. We have identified several AAV non-structural proteins which were previously undetected. At least one of these proteins is necessary for viral DNA replication. Post-translational processing does not account for production of any AAV structural proteins, although they share large proportions of sequences-in-common. It is now clear, however, that these proteins originate from independent in-frame initiations. The mechanism that regulates translation of AAV proteins is of fundamental interest, and we have now shown that one AAV structural protein is initiated by a codon not known previously to act as an initiation codon in eukaryotes. Furthermore, our current findings support a "scanning mechanism" in the translational expression of polycistronic eukaryotic mRNAs. Among methods used are affinity chromatography, gel electrophoresis, in vitro translation of viral RNA, electrophoretic and HPLC analyses of V8 protease and tryptic peptides and aminoterminal sequencing of purified polypeptides.